The long term goal of this project is to understand the molecular mechanism of the uridine-insertion/deletion type of RNA editing that occurs in the mitochondrion of kinetoplastid protozoa. The specific aims of this project are as follows: 1. Isolation of mitochondrial proteins and cloning of genes involved in RNA editing. Both enzymatic and structural proteins will be investigated. The genes will be cloned and expressed both in E. coli and as tagged proteins in L. tarentolae, and the recombinant proteins used to generate antibodies. Gene knockouts will be performed either by disruption of both alleles or by RNAi functional knockouts, and the effect on editing assayed both in vivo and in vitro. The recombinant proteins will be used to attempt to reconstitute editing activities in vitro. 2. Investigation of the mechanism of double strand RNA-induced (RNAi) inhibition of gene function. 3. Isolation of a functional 20S editing complex from Trypanosoma brucei procyclic cells; purification and identification of individual proteins and cloning genes. 4. Regulation of editing in T. brucei and Leishmania during life cycle, growth in culture, and during cell cycle. 5. Development of a transient mitochondrial expression system and a stable mitochondrial transformation system for use in studying RNA editing. 6. Investigation of the specific C34-U34 editing of the anticodon of imported tRNATrp in L. tarentolae. These parasites represent the causal agents of a variety of human animal and plant diseases, such as visceral and dermal leishmaniasis, Chagas Disease, African trypanosomiasis, and palm decay diseases. The existence of such a unique metabolic pathway as U-insertion/deletion RNA editing may provide a selective handle for chemotherapeutic intervention without affecting the host. Editing is required for the mt metabolism of these cells. A detailed knowledge of the enzymes involved in editing and the precise molecular pathways may allow the development of drugs which can kill the parasite and not affect the host.